CVB-SOP-0104.03 

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CONTROLLED//PROPIN//BASIC 

United States Department of Agriculture

Center for Veterinary Biologics 

Standard Operating Policy/Procedure 

Standard Bacterial Plate Count 

Date: March 15, 2024

Reference Number: CVB-SOP-0104.03

Contact: Center for Veterinary Biologics, 515-337-6100 

United States Department of Agriculture 

Animal and Plant Health Inspection Service 

P. O. Box 844 

Ames, IA 50010 

INTERNAL USE ONLY

Mention of trademark or proprietary product does not constitute a guarantee or warranty of the product by 

USDA and does not imply its approval to the exclusion of other products that may be suitable. 
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Table of Contents

1. Purpose

2. Materials

2.1 Equipment/instrumentation 

2.2 Reagents/supplies

3. Preparation for the Procedure

3.1 Personnel qualifications/training

3.2 Preparation of equipment, supplies, and reagents

3.3 Critical Control Points 

4. Performance of the Procedure 

5. Interpretation of Test Results 

6. Record and Report of Test Results

7. Summary of Revisions

Appendices
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1. Purpose

This document describes the titration methods used by the Bacteriology Section of the Center for 

Veterinary Biologics (CVB) to determine the colony-forming units (CFU) in final container 

samples. These methods use agar for determining CFUs and broths or solutions (see 

Appendices) as a diluent. 

2. Materials 

2.1 Equipment/instrumentation 

Equivalent equipment or instrumentation may be substituted for any brand name listed 

below. 

2.1.1 Vortex mixer

2.1.2 Colony counter

2.1.3 Pipetting aid

2.1.4 Incubator (Temperature and atmospheric conditions determined by 

bacterial species being cultured. See Appendices)

2.1.5 Laminar-flow Class II biosafety cabinet (BSC)

2.2 Reagents/supplies 

Equivalent reagents or supplies may be substituted for any brand name listed below. 

2.2.1 Diluent (see Appendices)

2.2.2 Plated agar media (see Appendices)

2.2.3 Reference culture(s) 

2.2.4 70% ethanol

2.2.5 Sterile water in serum vials

2.2.6 Inoculum spreader

2.2.7 Disposable syringes and needles

2.2.8 Sterile disposable pipettes 
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2.2.9 Sterile screw-capped culture tubes

2.2.10 Gloves and lab coat 

2.2.11 4 x 4-inch sterile gauze pads

2.2.12 Test tube rack 2.2.13 Sharps container 2.2.14 Micropipettors, 100-μL to 1.0-mL 

2.2.15 Pipette tips, 100-μL to 1-mL 

2.2.16 Pipetting aid 

2.2.17 Candle jar or appropriate anaerobic container

 3. Preparation for the Procedure 

3.1 Personnel qualifications/training 

Personnel must have experience or training in this protocol. This includes knowledge of 

aseptic biological laboratory techniques and preparation, proper handling, and disposal of 

biological agents, reagents, tissue culture samples, and chemicals. Personnel must also 

have knowledge of safe operating procedures and policies, as well as training in the 

operation of the necessary laboratory equipment listed in Section 2.1. 3.2 Preparation of equipment, supplies, and reagents 

3.2.1 Sterilize all glassware before use. 

3.2.2 Use only sterile bacteriological supplies (pipettes, syringes, needles, 

diluents, etc.). 

3.2.3 Operate all equipment and instrumentation according to manufacturer’s 

instructions and maintain according to standard operating procedures (SOPs). 

3.2.4 Turn on the BSC at least 30 minutes prior to use. 

3.2.5 Monitor temperature of incubators and freezers according to SOPs. 
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3.2.6 Label all plates with sample number or name, vial number, and dilution 

series. Label 2 or 3 (refer to the firm’s Outline of Production (OP)) plates per 

dilution series for each serial.

3.2.7 Warm the samples and reference culture to 20°-25°C before rehydrating to 

the appropriate volume. 

3.2.8 Store plates used for making counts at refrigerator temperature. Place 

plates to be used for counts in a 35°± 2°C incubator overnight prior to use or dry 

in a BSC before use. At the time of use, plates are no more than 3 months old. 

3.2.9 Prepare reference or positive controls according to the manufacturer’s 

instructions. 

3.3 Critical Control Points 

 3.3.1 All bacterial suspensions must be mixed well prior to drawing an aliquot 

for each subsequent serial dilution. 

3.3.2 All bacterial suspensions must be mixed well prior to placing an aliquot on 

an agar plate. 

3.3.3 Inoculum must be spread evenly on the surface of the agar plates and not 

allowed to pool around the edges. 

3.3.4 Do not use plates with fewer than 30 colonies (unless specified in the 

firm’s OP) as estimates for calculating CFU/dose or CFU/mL. 

3.3.5 

4. Performance of the Procedure 

4.1 Remove 2 vials (or the number of vials stated in the OP for testing) of product to 

be tested and 1 vial of reference stock culture from the freezer or cooler storage and allow 

to warm to 20°-25°C.

4.2 Disinfect the cap with 70% ethanol. Rehydrate the vials and allow the contents to 

reconstitute for at least 5 minutes. Shake the vials by inversion until thoroughly mixed.

4.3 Prepare a tenfold dilution series of the product by setting up a rack of 20 x 

150-mm screw-capped tubes and pipetting 9.0 mL of the diluent into each tube using a 

10-mL pipette. Label the tubes 10-1 to 10-x as needed.
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4.4 Transfer 1.0 mL of the first sample from Section 4.2 into the first tube of diluent 

by using a pipette or micropipettor with pipet tip. Cap the tube and vortex. The dilution 

series is continued by using a new pipette to transfer a 1.0 mL sample from this tube to 

the tube labeled 10-2. Repeat this method using a sterile pipette for each transfer until the 

required number of serial tenfold dilutions (refer to the firm’s OP) is attained. 

4.5 Deposit 0.1 mL* of the sample from the last 3 dilution points of the dilution series 

for the product onto the surface of media (see Appendices) using a sterile pipette or 

micropipettor with pipet tip. 

*Sometimes it is acceptable to plate larger amounts of samples onto the surface of 

the agar. This is dependent on the test and the firm’s OP. 

 4.6 Use a sterile inoculum spreader to evenly distribute the inoculum over the surface 

of the agar medium. 

4.6.1. The same inoculum spreader may be used on plates containing the same 

dilution. Change spreaders for each dilution plated. 

4.6.2. Avoid spreading inoculum completely to the edges of the plate. Inoculum 

may pool at the edges, resulting in colony growth that is difficult to quantitate.

4.7 Repeat Sections 4.4 through 4.6 with each of the rest of the vials of product. 

 4.8 Prepare 3 plates of media as in Sections 4.5 through 4.6 from each of 3 reference 

control dilutions as determined from Section 3.2.9. 

 4.9 Invert all plates and incubate according to the specifications in Appendix I. After 

incubation, count plates from each series that contains 30 to 300 CFUs**. Determine the 

mean CFU/mL or CFU per dose for the number of vials tested using the calculation(s) 

listed below.

**Exceptions to the dilution that yields 30-300 colonies per plate can be found in the 

firm’s OP. 

(Average count) = CFU/mL 

(Dilution plated) (mL plated) 

 

 or

 (Average Count) x (mL used to rehydrate) = CFU/dose

 (Dilution used) x (mL plated) x (Number of doses) 

or 
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(Average count) (mL per animal) (Challenge dilution) = CFU/dose 

 (Dilution plated) (mL plated) (Number of doses) 

Example: Triplicate plates inoculated with 0.1 mL of a 10-5 dilution of 

culture yielded counts of 82, 79, and 88 colonies, respectively. 

Average number of colonies = 82+79+88 = 83 CFU 

3

83 CFU x 1 x 1 = 8.3 x 107 CFU/mL

 10-5 0.1 mL 

4.10 If more than one dilution series has valid CFUs, determine the geometric mean 

CFU per dose for the number of vials tested using the calculation(s) listed below. 

Note: Because of the logarithm and exponentials, rounding error can get easily 

magnified. For the final result to be accurate to two decimal places, use at least 

three decimal places during the calculation. 

Example: 

In vial 1, triplicate plates inoculated with 0.1 mL of a 10-5 dilution of culture 

yielded counts of 82, 79, and 88 colonies, respectively. 

Average number of colonies = 82+79+88 = 83 CFU 

3

 (83 CFU) x (20 mL) = 1660 CFU/mL = 8.3 x 107 CFU/dose

 (10-5) x (0.1 mL) x (20 doses) 2 x 10-5 mL/dose 

 In vial 2, triplicate plates inoculated with 0.1 mL of a 10-6 dilution of 

culture yielded counts of 102, 109, and 108 colonies, respectively. 

Average number of colonies = 102+109+108 = 106 CFU

3

 (106 CFU) x (20 mL) = 2120 CFU/mL = 1.06 x 109 CFU/dose

 (10-6) x (0.1 mL) x (20 doses) 2 x 10-6 mL/dose 

Geometric mean CFU/dose of 2 vials = 10 ( . × / ) ( . × / )

= 2.97 x 108 CFU/dose 

 

 
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5. Interpretation of Test Results 

5.1 If on the initial test the CFU per dose is equal to or exceeds the required minimum 

as written in the firm’s OP, the serial or subserial is satisfactory (SAT) for bacterial count 

without additional testing. 

5.2 If on the initial test the CFU per dose is less than the required minimum release

titer as written in the firm’s OP, the serial or subserial may be retested (RT) using double 

the amount of new vaccine samples used in the initial test, provided that if the retest is 

not done, the serial or subserial is unsatisfactory (UNSAT). Compare the firm’s OP 

method to this SOP when retesting with the new vaccine samples. If on the RT, the

average count of the new vaccine samples with the firm’s OP method is less than the 

required minimum, the serial or subserial is UNSAT.

5.3 If on the retest with the new vaccine samples, the average using the firm’s OP 

method count is equal to or exceeds the required minimum, the serial is SAT. 5.4 If on the initial test, the reference culture or positive control culture is not within 

the titer range determined in Section 3.2.9, but the serial being tested has a SAT result, 

the serial or subserial is a no test (NT) for bacterial count without additional testing and 

the product is released on the results of the firm’s tests. If the reference culture is not 

within the titer and the serial being tested is below the minimum release titer, the serial is 

retested without bias using the same number of new vaccine samples as the initial test. If 

on the initial test there is growth on the negative control plates, the serial or subserial is a 

NT for bacterial count without additional testing. 

6. Record and Report of Test Results 

Record and report results of the test(s) according to SOPs. 

7. Summary of Revisions 

Version CVB-SOP-0104.03 

Updated cover page and reformatted document.

Version CVB-SOP-0104.02 

Alphanumeric number changed from BBSOP0019.04 to CVB-SOP-0104.02. 

The contact has been updated. 

4.10: Added to clarify calculation practices for multiple dilution series with valid 

CFUs. 
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CONTROLLED//PROPIN//BASIC 

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Updated room temperature to 20°-25°C in Sections 3.7 and 4.1.

Version BBSOP0019.04

The Bacteriology Section Leader has been updated. 

Sections 1-7: These sections have been rewritten for clarification and updated to 

reflect current practices. 

Appendix II: Media storage conditions have been updated.

Version BBSOP0019.03

Appendix II.6: Ethyl alcohol has been changed to QH2O.

Version BBSOP0019.02 

The Contact has been changed from Nancy Clough to Sophia Campbell and Amanda 

Byersdorfer. 

4.1: Equipment and supplies have been added.

4.2: This section has been updated to reflect current practices.

4.3: This section has been updated to reflect current practices.

7: This section has been added to explain interpretation of test results for firms’ 

vaccines.

Appendix I: This has been added to list media, media numbers, incubation 

temperature, incubation time, and media formulations.

Appendix II: Media storage information has been added. 
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Appendix I 

Media, Incubation Temps/Duration 

(Or as stated in the Manufacturer’s Outline of Production) 

1

for the National Centers for Animal Health (NCAH) media formulations, see Appendix II

Organism Agar *NCAH 

Media 

Number

Diluent 

1NCAH

Media 

Number

Incubation

Temperature 

Duration of 

Incubation 

Salmonella 

cholerasuis 

Trypticase 

Soy Agar 

w/5% Bovine 

Blood 

10205 Tryptose 

Phosphate 

Broth 

10426 35°± 2°C Up to 48 

hours 

Escherichia

coli 

Trypticase 

Soy Agar 

10487 Tryptose 

Phosphate 

Broth

10426 35°± 2°C 24 hours 

Pasteurella 

haemolyticamultocida 

Trypticase 

Soy Agar 

w/5% Sheep 

Blood & 

Additives 

10596 MuellerHinton 

Broth 

10225 35°± 2°C Up to 96 

hours 

Pasteurella 

anatipestifer 

Trypticase 

Soy Agar w / 

0.05% Yeast 

Extract 

10225 Trypticase 

Soy Broth 

10423 35°± 2°C Up to 48 

hours 

anaerobicall

y

Bordetella 

bronchiseptica 

BordetGengou agar 

with 15% 

ovine blood

10157 1% Peptone 

SalineSolution 

10138 35°± 2°C Up to 72 

hours 

Bordetella 

avium 

Tryptose 

Agar w/ 5% 

Fetal Bovine 

Serum

10218 Tryptose 

Broth 

10404 35°± 2°C Up to 72 

hours 

Streptococcus 

equi 

Columbia 

CNA Agar 

w/5% Sheep 

Blood 

10671 1% Peptone 

Saline 

Solution 

10138 35°± 2°C Up to 48 

hours 

Salmonella 

dublin 

Trypticase 

Soy Agar 

10487 1% Peptone 

Saline 

Solution

10138 35°± 2°C Up to 48 

hours 

Salmonella 

typhimurium 

Blood Agar 

Base with 5% 

Bovine 

Blood 

10006 Trypticase 

Soy Broth 

10423 35°± 2°C Up to 48 

hours 

Edwardsiella 

ictaluri 

Trypticase 

Soy Agar 

w/5% Sheep 

Blood 

10210 1% Peptone 

Saline 

Solution 

10138 25°± 2°C 48 hours in 

humidified 

atmosphere 
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Actinobacillus 

pleuropneumo

niae 

BHIA with 

5% NAD 

10697 BHI broth 

with NAD 

and 

dextrose

10347 35°± 2°C 48 hours 

Haemophilus 

parasuis

Chocolate 

Agar II w/ 

Hemoglobin 

and 

Isovitalex 

10598 PPLO 

Broth w/o 

CV w/ 

Additives + 

400 μg/mL 

Streptomyci

n sulfate

10600 36°± 3°C 24-72 hours 

anaerobicall

y or in 4-6% 

CO2

Flavobacteriu

m columnare 

Modified 

Cytophaga 

Agar 

11012 Tryptone 

Water 

w/1% 

Peptone 

10365 27°- 29°C 60-120 

hours 

humidified 

atmosphere 

Arthrobacter 

spp. 

Modified 

Tryptic Soy 

Agar

10278 0.9% 

Sodium 

Chloride 

30192 22°C 5-7 days
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Appendix II 

Media Formulations 

1. Tryptose phosphate broth, NCAH Media #10426 

Tryptose phosphate broth (Difco) 29.5 g 

H2O 1000.0 mL 

Autoclave 20 minutes at 121°C. Store at 2°- 7°C for up to 3 months. 

2. Trypticase soy agar with 5% bovine blood (defibrinated), NCAH Media #10205 

Trypticase soy agar (BBL) 40.0 g 

QH2O 950.0 mL 

Autoclave 20 minutes. Cool to 47°C in waterbath. Add: defibrinated bovine blood, 50.0 mL. 

Store at 2°- 7°C for up to 3 months. 

3. Trypticase soy agar (TSA), NCAH Media #10487

Trypticase soy agar (BBL) 40.0 g 

QH2O 1000.0 mL 

Autoclave 20 minutes at 121°C. Store at 2°- 7°C for up to 3 months.

4. Mueller-Hinton broth, NCAH Media #10225

Mueller Hinton broth 21.0 g 

QH2O 1000.0 mL

Gently warm to dissolve and autoclave 15 minutes at 121°C. Store at 2°- 7°C for up to 3 months. 

Add 2.5 mL each of 1% yeast, supplement B, and 10% streptomycin solution before using in a 

test. Dispose of unused complete broth after use.

5. 1% Yeast Solution, NCAH Media #10999

Yeast extract (Difco) 1.0 g

QH2O 99.0 mL 

Gently warm to dissolve and autoclave 15 minutes at 121°C. Store at 2°- 7°C for up to 3 months. 
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6. 10% Streptomycin Solution, NCAH Media #10999

Dihydrostreptomycin 10.0 g 

QH2O 90.0 mL

Filter and dispense. 

7. Bacto-Supplement B with Bacto-Supplement reconstituting fluid, 6 x 10 mL, cat. 

#0276-60-4, Difco Laboratories, Detroit, MI 48232 

8. Trypticase soy agar w/ 5% sheep blood w/ additives, NCAH Media #10596

Trypticase soy agar 40.0 g 

QH2O 939.0 mL 

QS for blood and additives. Mix and autoclave 20 minutes at 121°C. Cool to 56°C in 

waterbath. 

Add: 

Defibrinated sheep blood 50.0 mL 

1% yeast extract (Difco) powder 6.25 mL 

10% streptomycin 5.0 mL 

Yeast and streptomycin can be made in stock solutions and stored in refrigerator. Store 

completed agar at 2°- 7°C for up to 3 months. 

9. Trypticase soy broth (TSB), NCAH Media #10423 

Trypticase soy broth 30.0 g 

H2O 1000.0 mL

Autoclave 20 minutes at 121°C. Store at 2°- 7°C for up to 3 months. 

10. Trypticase soy agar w/ 0.05% yeast extract, NCAH Media #10543

Trypticase soy agar 40.0 g

Yeast extract 0.5 g

QH2O 1000.0 mL 

121°C. Cool in 46°- 50°C water bath. Store at 2°- 7°C for up to 3 

months.
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11. Peptone solution 1% + 0.5% NaCl, NCAH Media #10138 

Bacto peptone 10.0 gm 

NaCl 5.0 gm 

QH2O 1000.0 mL 

Autoclave for 20 minutes at 121°C. Store at 2°- 7°C for up to 3 months. 

12. Bordet-Gengou agar with 15% sheep blood (defibrinated), NCAH Media #10157 

Bordet-Gengou agar 30.0 gm 

Glycerol 10.0 mL 

H2O QS to 840.0 mL

Heat to boiling. Autoclave for 15 minutes. Cool. Add 150 mL of defibrinated sheep blood. Store 

at 2°- 7°C for up to 3 months. 

13. Tryptose broth, NCAH Media #10404 

Tryptose broth 26.0 g 

H2O 1000.0 mL 

Autoclave 20 minutes at 121°C. Store at 2°- 7°C for up to 3 months. 

14. Tryptose agar with 5% fetal bovine serum, NCAH Media #10218

Tryptose agar 41.0 gm 

QH2O 950.0 mL 

Autoclave for 25 minutes. Cool in waterbath at 56°C and add 50 mL fetal bovine serum. Store at 

2°- 7°C for up to 3 months. 

15. Columbia CNA agar, NCAH Media #10671

Columbia CNA agar 42.5 g 

QH2O 950.0 mL 

Heat to boil for 1 minute. Autoclave 12 minutes at 121°C. Cool to 46°- 50°C. Add defibrinated 

sheep blood - 50.0 mL. Store at 2°- 7°C for up to 3 months. 
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16. Blood agar base with 5% bovine blood, NCAH Media #10006

Blood agar base (Difco) 40.0 g

H2O 950.0 mL

Autoclave 20 minutes. Cool to 47°C. Add defibrinated bovine blood - 50.0 mL. Store at 2°- 7°C 

for up to 3 months. 

17. Trypticase soy agar with 5% sheep blood (defibrinated), NCAH Media #10210 

Trypticase soy agar 40.0 g 

QH2O 950.0 mL 

121°C. Cool to 46°- 50°C in water bath. Add 

defibrinated sheep blood - 50.0 mL. Store at 2°- 7°C for up to 3 months. 

18. Brain heart infusion broth with NAD and dextrose, NCAH Media #10347 

Brain heart infusion broth 37.0 g 

QH2O 1000.0 mL 

Autoclave for 20 minutes at 121°C. 

Aseptically add: 

NAD, 5% solution 0.4 mL 

Dextrose solution, 50% 10.0 mL 

Mix and dispense as requested. Store at 2°- 7°C for up to 3 months. 

19. Brain heart infusion agar with 5% NAD, NCAH Media #10697 

Brain heart infusion agar 52.0 g 

NAD 5% solution (0.5 g/10.0 mL) 10.0 mL 

QH2O 990.0 mL 

Mix brain heart infusion agar in aspirator bottle and autoclave at 121°C for 20 minutes per L. 

Place in water bath and let cool to 50°C. This is critical. NAD will break down if agar is 

warmer than 50°C. 

Aseptically add 10.0 mL of filtered NAD and dispense as requested. Store at 2°- 7°C for up to 3 

months.
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20. Chocolate Agar II w/ Hemoglobin and Isovitalex, NCAH Media #10598

GC agar base (BBL) 21.6 g

Bacto agar 2.0 g 

QH2O 600.0 mL 

Mix in an aspirator jug. Set aside. 

Hemoglobin 10.0 g 

QH2O 400.0 mL 

Mix in a screw-capped flask. Autoclave both 20 minutes at 121°C. Cool in a 56° water bath. 

Aseptically combine the two solutions, and add:

Isovitalex 10.0mL 

Dispense as requested. Store at 2°- 7°C for up to 3 months.

21. PPLO Broth w/o CV w/ Additives + 400 μg/mL Streptomycin sulfate, NCAH Media 

#10600

PPLO broth w/o CV 21.0 g 

QH2O 1000.0 mL 

Mix together. Autoclave 15 minutes at 121°C. Cool to 56°C. Aseptically add: 

Horse serum (heat inactivated) 50.0 mL 

PPLO yeast extract (NCAH Media #40069) 100.0 mL 

Streptomycin sulfate 400.0 μg/mL 

Mix well. Dispense as requested. Store at 2°- 7°C for up to 3 months. 

22. Modified Cytophaga Agar, NCAH Media #11012

Typtone 10.0 g 

Yeast extract 0.5 g 

Beef extract 0.2 g 

Sodium acetate 0.2 g 

Super Q water 100.0 mL 

Combine ingredients, mix well. Adjust pH to 7.3. Add:

Bacto agar 9.0 g 
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Autoclave at 121°C. Dispense as requested. May also be autoclaved after dispensing. Store at 

2°- 7°C for up to 3 months. 

23. Tryptone Water w/ 1% Peptone, NCAH Media #10365

Tryptone 10.0 g 

Sodium chloride 5.0 g 

Peptone 10.0 g 

QH2O 1000.0mL 

Mix all ingredients and dispense as requested. Autoclave 15 minutes at 121°C. Store at 2°- 7°C 

for up to 3 months. 

24. Modified Tryptic Soy Agar, NCAH Media #10278

Tryptic soy broth 30.0 g 

Yeast extract 15.0 g 

Antifoam 1.5 mL/L 

RO water Q.S. to 1000.0 mL 

Agar 20.0 g 

Combine, mix well. Autoclave at 121°C for 15 minutes. Store at 2°- 7°C for up to 3 months. 

25. 0.9% Sodium Chloride, NCAH Media #30192 

Sodium chloride 9.0 g 

Q H2O 1000.0 mL 

Mix and autoclave 20 minutes at 121°C. Store at 2°- 7°C for up to 3 months.